Instituto de Investigaciones Biológicas Clemente Estable

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.

ABSTRACT Sandy beaches line most of the world's oceans and are highly valued by society: more people use sandy beaches than any other type of shore. While the economic and social values of beaches are generally regarded as paramount, sandy shores also have special ecological features and contain a distinctive biodiversity that is generally not recognized. These unique ecosystems are facing escalating anthropogenic pressures, chiefly from rapacious coastal development, direct human uses — mainly associated with recreation — and rising sea levels. Beaches are increasingly becoming trapped in a ‘coastal squeeze’ between burgeoning human populations from the land and the effects of global climate change from the sea. Society's interventions (e.g. shoreline armouring, beach nourishment) to combat changes in beach environments, such as erosion and shoreline retreat, can result in severe ecological impacts and loss of biodiversity at local scales, but are predicted also to have cumulative large‐scale consequences worldwide. Because of the scale of this problem, the continued existence of beaches as functional ecosystems is likely to depend on direct conservation efforts. Conservation, in turn, will have to increasingly draw on a consolidated body of ecological theory for these ecosystems. Although this body of theory has yet to be fully developed, we identify here a number of critical research directions that are required to progress coastal management and conservation of sandy beach ecosystems.

Two microsporidia species have been shown to infect Apis mellifera, Nosema apis and Nosema ceranae. This work presents evidence that N. ceranae infection significantly suppresses the honey bee immune response, although this effect was not observed following infection with N. apis. Immune suppression would also increase susceptibility to other bee pathogens and senescence. Despite the importance of both Nosema species in honey bee health, there is no information about their effect on the bees' immune system and present results can explain the different virulence between both microsporidia infecting honeybees.

Despite aging being by far the greatest risk factor for highly prevalent neurodegenerative disorders, the molecular underpinnings of age-related brain changes are still not well understood, particularly the transition from normal healthy brain aging to neuropathological aging. Aging is an extremely complex, multifactorial process involving the simultaneous interplay of several processes operating at many levels of the functional organization. The buildup of potentially toxic protein aggregates and their spreading through various brain regions has been identified as a major contributor to these pathologies. One of the most striking morphologic changes in neurons during normal aging is the accumulation of lipofuscin (LF) aggregates, as well as, neuromelanin pigments. LF is an autofluorescent lipopigment formed by lipids, metals and misfolded proteins, which is especially abundant in nerve cells, cardiac muscle cells and skin. Within the Central Nervous System (CNS), LF accumulates as aggregates, delineating a specific senescence pattern in both physiological and pathological states, altering neuronal cytoskeleton and cellular trafficking and metabolism, and being associated with neuronal loss, and glial proliferation and activation. Traditionally, the accumulation of LF in the CNS has been considered a secondary consequence of the aging process, being a mere bystander of the pathological buildup associated with different neurodegenerative disorders. Here, we discuss recent evidence suggesting the possibility that LF aggregates may have an active role in neurodegeneration. We argue that LF is a relevant effector of aging that represents a risk factor or driver for neurodegenerative disorders.

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the 'MiDAS 4' database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.

The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.

Primary cultures of rat embryonic motor neurons deprived of brain-derived neurotrophic factor (BDNF) induce neuronal nitric oxide synthase (NOS) within 18 hr. Subsequently, >60% of the neurons undergo apoptosis between 18 and 24 hr after plating. Nitro-L-arginine and nitro-L-arginine methyl ester (L-NAME) prevented motor neuron death induced by trophic factor deprivation. Exogenous generation of nitric oxide at concentrations lower than 100 nM overcame the protection by L-NAME. Manganese tetrakis (4-benzoyl acid) porphyrin, a cell-permeant superoxide scavenger, also prevented nitric oxide-dependent motor neuron death. Motor neurons cultured without trophic support rapidly became immunoreactive for nitrotyrosine when compared with motor neurons incubated with BDNF, L-NAME, or manganese TBAP. Our results suggest that peroxynitrite, a strong oxidant formed by the reaction of NO and superoxide, plays an important role in the induction of apoptosis in motor neurons deprived of trophic factors and that BDNF supports motor neuron survival in part by preventing neuronal NOS expression.

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1(G93A) rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1(G93A) astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1(G93A) astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1(G93A) rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1(G93A) astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1(G93A) astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (approximately 10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1(G93A) astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.

Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (> 2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 microM) intially (3-4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.

The PREDICTS project-Projecting Responses of Ecological Diversity In Changing Terrestrial Systems (www.predicts.org.uk)-has collated from published studies a large, reasonably representative database of comparable samples of biodiversity from multiple sites that differ in the nature or intensity of human impacts relating to land use. We have used this evidence base to develop global and regional statistical models of how local biodiversity responds to these measures. We describe and make freely available this 2016 release of the database, containing more than 3.2 million records sampled at over 26,000 locations and representing over 47,000 species. We outline how the database can help in answering a range of questions in ecology and conservation biology. To our knowledge, this is the largest and most geographically and taxonomically representative database of spatial comparisons of biodiversity that has been collated to date; it will be useful to researchers and international efforts wishing to model and understand the global status of biodiversity.

Polyhydroxyalkanoates (PHAs) are a group of carbon andenergy storage compounds that are accumulated during suboptimal growth by many bacteria, and intracellularly deposited in the form of inclusion bodies. Accumulation of PHAs is thought to be used by bacteria to increase survival and stress tolerance in changing environments, and in competitive settings where carbon and energy sources may be limited, such as those encountered in the soil and the rhizosphere. Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial ecology, and holds great potential for the improvement of bacterial inoculants for plants and soils. This review summarizes the current knowledge on the ecological function of PHAs, and their strategic role as survival factors in microorganisms under varying environmental stress is emphasized. It also explores the phylogeny of the PHA cycle enzymes, PHA synthase, and PHA depolymerase, suggesting that PHA accumulation was earlier acquired and maintained during evolution, thus contributing to microbial survival in the environment.

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species' threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project - and avert - future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups - including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems - http://www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015.

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.

In the past centuries, viruses have benefited from globalization to spread across the globe, infecting new host species and populations. A growing number of viruses have been documented in the western honey bee, Apis mellifera. Several of these contribute significantly to honey bee colony losses. This review synthetizes the knowledge of the diversity and distribution of honey-bee-infecting viruses, including recent data from high-throughput sequencing (HTS). After presenting the diversity of viruses and their corresponding symptoms, we surveyed the scientific literature for the prevalence of these pathogens across the globe. The geographical distribution shows that the most prevalent viruses (deformed wing virus, sacbrood virus, black queen cell virus and acute paralysis complex) are also the most widely distributed. We discuss the ecological drivers that influence the distribution of these pathogens in worldwide honey bee populations. Besides the natural transmission routes and the resulting temporal dynamics, global trade contributes to their dissemination. As recent evidence shows that these viruses are often multihost pathogens, their spread is a risk for both the beekeeping industry and the pollination services provided by managed and wild pollinators.

The grasslands of southeastern South America (SESA), comprising one of the most extensive grassland ecosystems in the Neotropics, have been negatively impacted by the development of the livestock industry, arable agriculture, and forestry. SESA grasslands have a rich avifauna that includes 22 globally threatened and near-threatened species, and many other species have suffered local population extinctions and range reductions. In addition to habitat loss and fragmentation, grassland birds in SESA are threatened by improper use of agrochemicals, unfavorable fire management regimes, pollution, and illegal capture and hunting. Studies to date have provided information about the distribution of grassland birds, the threats populations face, and the habitat requirements of some threatened species, but more information is needed concerning dispersal and migration patterns, genetics, and factors that influence habitat use and species survival in both natural and agricultural landscapes. There are few public protected areas in the region (1% of original grasslands), and many populations of threatened grassland birds are found on private lands. Therefore, efforts to preserve grassland habitat must reconcile the interests of land owners and conservationists. Current conservation efforts include establishment of public and private reserves, promotion of agricultural activities that reconcile production with biodiversity conservation, development of multilateral conservation projects across countries, and elaboration of action plans. Measures that result in significant losses to private land owners should include economic compensation, and use of economic incentives to promote agriculture and forestry in native grassland areas should be discouraged, especially in priority areas for grassland birds. Although more studies are needed, some actions, particularly habitat protection and improved management of public and private lands, should be taken immediately to improve the conservation status of grassland birds in SESA.

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.